Patients with ovarian cancer are typically diagnosed at an advanced stage, resulting in poor prognosis since there are currently no effective early-detection screening tests for women at average-risk for ovarian cancer. Here, we investigated the effects of MT-6, a derivative of moscatilin, in ovarian cancer cells. Our investigation showed that MT-6 inhibited the proliferation and viability of ovarian cancer cells with submicromolar IC50 values. MT-6&ndash;treated SKOV3 cells showed significant cell cycle arrest at G2/M phase, followed by an increase in the proportion of cells in a sub-G1 phase. In addition, MT-6 induced a concentration-dependent increase in mitotic markers, mitotic kinases, cell cycle regulators of G2/M transition, and apoptosis-related markers in ovarian cancer cells. MT-6 treatment also induced mitochondrial membrane potential loss, JNK activation, and DR5 expression. Cotreatment of cells with the JNK inhibitor SP600125 considerably attenuated MT-6&ndash;induced apoptosis, mitochondria membrane potential loss, DR5 upregulation, and suppression of cell viability. MT-6 also inhibited tumor growth in an SKOV3 xenograft model without significant body weight loss. Together, our findings suggest that MT-6 is a potent anticancer agent with tumor-suppressive activity in vitro and in vivo that could be further investigated for ovarian cancer therapy in the future.

f6: MT-6–induced Δψm loss and mitotic arrest are JNK dependent.(A,B) MT-6 upregulates p-JNK levels in a concentration- (A) and time- (B) dependent manner. Effects of MT-6 on survival signaling were investigated by Western blot analysis. (C) MT-6–induced mitotic arrest was reversed by cotreatment with the JNK inhibitor SP600125. SKOV3 cells were treated with the indicated concentrations of MT-6 for the indicated times, with or without SP600125 cotreatment, then whole-cell lysates were analyzed by Western blotting. (D) Cotreatment with the JNK inhibitor SP600125 substantially attenuates MT-6–induced Δψm loss. Cells were treated with 0.3 μM MT-6 for 24 h, then Δψm was measured by rhodamine 123 (10 μM) staining followed by flow cytometry analysis.

Mentions:
To further investigate the mechanism of MT-6–induced cytotoxicity and cell-cycle dysregulation, we used Western blotting to examine several kinases that have pro-survival activities or play roles in regulating the cell cycle. These experiments revealed that MT-6 induced a significant concentration- and time-dependent reduction in p-Akt and p-ERK levels, and increased p-JNK (Fig. 6A and B). Previous studies have reported that activation of JNK plays an important role in mitotic arrest, leading to inhibition of cancer development and apoptosis3132. Cotreatment of cells with the JNK inhibitor, SP600125, significantly reversed MT-6–induced upregulation of mitotic arrest markers and Δψm loss (Fig. 6C and D), indicating that JNK plays a crucial role in MT-6–induced cell cycle dysfunction and Δψm loss. In addition, cotreatment with SP600125 also attenuated MT-6–induced cytotoxicity (Fig. 7A), accumulation of a subG1 population (Fig. 7B), and expression of apoptosis-associated proteins (Fig. 7C). Inactivation of JNK by siRNA also reversed MT-6-increased cell mitotic arrest and apoptosis (Supplementary Fig. S2). Moreover, cotreatment with SP600125 counteracted DR5 induction by MT-6 treatment, suggesting that MT-6 activates the JNK-DR5-apoptosis pathway.

f6: MT-6–induced Δψm loss and mitotic arrest are JNK dependent.(A,B) MT-6 upregulates p-JNK levels in a concentration- (A) and time- (B) dependent manner. Effects of MT-6 on survival signaling were investigated by Western blot analysis. (C) MT-6–induced mitotic arrest was reversed by cotreatment with the JNK inhibitor SP600125. SKOV3 cells were treated with the indicated concentrations of MT-6 for the indicated times, with or without SP600125 cotreatment, then whole-cell lysates were analyzed by Western blotting. (D) Cotreatment with the JNK inhibitor SP600125 substantially attenuates MT-6–induced Δψm loss. Cells were treated with 0.3 μM MT-6 for 24 h, then Δψm was measured by rhodamine 123 (10 μM) staining followed by flow cytometry analysis.

Mentions:
To further investigate the mechanism of MT-6–induced cytotoxicity and cell-cycle dysregulation, we used Western blotting to examine several kinases that have pro-survival activities or play roles in regulating the cell cycle. These experiments revealed that MT-6 induced a significant concentration- and time-dependent reduction in p-Akt and p-ERK levels, and increased p-JNK (Fig. 6A and B). Previous studies have reported that activation of JNK plays an important role in mitotic arrest, leading to inhibition of cancer development and apoptosis3132. Cotreatment of cells with the JNK inhibitor, SP600125, significantly reversed MT-6–induced upregulation of mitotic arrest markers and Δψm loss (Fig. 6C and D), indicating that JNK plays a crucial role in MT-6–induced cell cycle dysfunction and Δψm loss. In addition, cotreatment with SP600125 also attenuated MT-6–induced cytotoxicity (Fig. 7A), accumulation of a subG1 population (Fig. 7B), and expression of apoptosis-associated proteins (Fig. 7C). Inactivation of JNK by siRNA also reversed MT-6-increased cell mitotic arrest and apoptosis (Supplementary Fig. S2). Moreover, cotreatment with SP600125 counteracted DR5 induction by MT-6 treatment, suggesting that MT-6 activates the JNK-DR5-apoptosis pathway.

Patients with ovarian cancer are typically diagnosed at an advanced stage, resulting in poor prognosis since there are currently no effective early-detection screening tests for women at average-risk for ovarian cancer. Here, we investigated the effects of MT-6, a derivative of moscatilin, in ovarian cancer cells. Our investigation showed that MT-6 inhibited the proliferation and viability of ovarian cancer cells with submicromolar IC50 values. MT-6&ndash;treated SKOV3 cells showed significant cell cycle arrest at G2/M phase, followed by an increase in the proportion of cells in a sub-G1 phase. In addition, MT-6 induced a concentration-dependent increase in mitotic markers, mitotic kinases, cell cycle regulators of G2/M transition, and apoptosis-related markers in ovarian cancer cells. MT-6 treatment also induced mitochondrial membrane potential loss, JNK activation, and DR5 expression. Cotreatment of cells with the JNK inhibitor SP600125 considerably attenuated MT-6&ndash;induced apoptosis, mitochondria membrane potential loss, DR5 upregulation, and suppression of cell viability. MT-6 also inhibited tumor growth in an SKOV3 xenograft model without significant body weight loss. Together, our findings suggest that MT-6 is a potent anticancer agent with tumor-suppressive activity in vitro and in vivo that could be further investigated for ovarian cancer therapy in the future.